Oral Presentation ESA-SRB-NZSE-CaSR 2026 in conjunction with ENSA

Blastomere separation beyond the 8-cell stage enables bovine embryo multiplication and viable calf production (143985)

Danielle Azoulay 1 , Nicholas I. Cameron 2 , Wilhelmus T.L. te Brake 2 , Michael B. Morris 1 , Margot L. Day 1
  1. School of Medical Sciences , The University of Sydney, Sydney, NSW, Australia
  2. Nbryo Pty Ltd, Brisbane, QLD, Australia

Background:

Embryo multiplication by blastomere separation can increase embryo output by generating multiple embryos from one zygote (1). In cattle, blastocysts have been generated from blastomeres isolated before the 8-cell stage, but multiplication has not exceeded four embryos per original embryo (2-5). Later pre-compaction stages represent an unexplored opportunity because they contain more blastomeres and have progressed beyond the 8-cell developmental block, a major bottleneck in bovine in vitro-production.

Methods:

A culture platform for zona pellucida-free blastomeres was developed using round-bottom wells coated with bovine serum albumin or polyvinyl alcohol (PVA). Blastomeres isolated from Day 4 8-32-cell stage embryos were cultured individually or aggregated as pairs or quadruplets. Blastocyst formation was assessed on Day 7, and total cell number and lineage allocation were evaluated by SOX2 (inner cell mass, ICM) and CDX2 (trophectoderm) immunostaining. Embryo transfer was used to assess pregnancy establishment and calving. Proportional and cell-number data were analysed using chi-square tests and unpaired t-tests, respectively.

Results:

Both coatings prevented blastomere adhesion and supported development, with PVA selected as a chemically defined surface. Blastocyst formation from individual 8-cell stage blastomeres was highest (19.9%), followed by 16-cell (13.6%) and 32-cell (3.5%) stages. Quadruplets of 32-cell-stage blastomeres achieved the highest blastocyst formation rate (61.4%), compared with pairs from the 8-cell (26.6%), 16-cell (47.2%) and 32-cell (29.1%) stages. Pairs of 16-cell-stage blastomeres and quadruplets of 32-cell-stage blastomeres yielded 3.9 and 4.9 blastocysts per original embryo, respectively. Both produced SOX2+ and CDX2+ cells with more total cells in quadruplet-derived verses pair-derived blastocysts (32.7±2.1 vs 23.0±1.9), whereas ICM cell number did not differ. Both strategies established pregnancies and produced live offspring, with 38 calves generated.

Conclusion:

This study demonstrated for the first time that bovine blastomere separation beyond the 8-cell stage can generate multiple blastocysts from a single embryo and produce live offspring.

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